Immunoreactive peptide and method of diagnosing rheumatoid arthritis using said peptide

FIELD: chemistry.

SUBSTANCE: invention relates to peptides which originate from an antigen recognised by autoantibodies used for diagnosing rheumatoid arthritis. The peptides are filaggrin molecule fragments which contain modified residues of arginine and having amino acid sequences given in the formula of invention. The invention discloses a method of diagnosing rheumatoid arthritis by detecting autoimmune antibodies using the said peptide(s) through reaction of the latter with the blood serum of patients suffering from rheumatoid arthritis. Presence of autoimmune antibodies in the analysed sample is indicated by presence of peptide complexes formed with the antibody.

EFFECT: disclosed peptide has high specificity and sensitivity.

4 cl, 1 dwg, 3 tbl


The invention relates to peptides derived from the antigen recognized by autoantibodies. The peptides are fragments of molecules filaggrin containing modified arginine residues. The invention relates also to method of detection of autoimmune antibodies in rheumatoid arthritis by interacting with autoimmune antibodies from patients suffering from rheumatoid arthritis. Rheumatoid arthritis (RA) is one of the most common human diseases. This disease according to who suffers 1-2% of the population. In Russia officially registered more than 1 million patients with RA. RA is characterized by chronic inflammation of the joints. This leads to suffering, reduced quality of life, reduce disability.

The development of RA is characterized by an extraordinary diversity of clinical manifestations and biochemical disorders. This significantly hinders early diagnosis and, therefore, the provision of adequate therapy, which is especially effective at the onset of the disease. Modern diagnosis of RA is based on clinical manifestations of the disease in combination with laboratory determination in the patient's blood so-called rheumatoid factor (RF). However, RF is not sufficiently specific marker. Two other serological markers of RA are antiperiodic the RNA factor (APF) and anticarcinoma antibodies (AKA), recognize antigenic determinants citrullinaemia protein filaggrin. Despite the high specificity of the APF and AKA for the diagnosis of RA, these autoantibodies are not widely used in clinical laboratory practice due to technical difficulties. In recent years there has been developed enzyme-linked immunosorbent (ELISA) method for the determination of antibodies to filaggrin (AF), which has a high specificity. AF found in the blood before the onset of clinical signs of RA, which demonstrates the important role of these antibodies in the pathogenesis of this disease and the ability to conduct early diagnosis for this indicator. The basis of this technique is antigenic complex in the form of polypeptides (sections molecules filaggrin) or the protein (recombinant).

From the prior art known peptides used in the method for the diagnosis of rheumatoid arthritis and described US 2004241767, US 2004253644, US 6890720, US 7288634, US 6858438.

The invention dedicated to the creation of new antigenic complex, with greater specificity and sensitivity.

According to theory of the heterogeneity of the population AF, reflecting the natural variability of antigens to which they are produced (the presence of ACE, AKA, AF), and polymorphism molecules filaggrin we can assume that there are several carolinecaroline structural participants who s this molecule, possessing antigenic properties. Computer analysis of molecules confirmed it.

Based on predictive analysis of antigenic epitopes solid-phase method using f-moc equipment was synthesized four peptide reproducing fragments of protein molecules filaggrin: f-1, f-2, f-3, f-4.





where X is Cit,

which is immunologically reactive immunological study aimed at identifying RA, and can be used in the method for the diagnosis of RA, including the detection of autoimmune antibodies in the serum of the patient by interaction of at least one of the foregoing peptides from serum of a patient and detecting the presence of the formed complexes between the peptide and an antibody.

All the preparations of peptides corresponded to 95% purity according to HPLC and mass spectroscopy and contained 20 amicalola with the replacement of two Arg residues at Cit.

ELISA-methods of measurement of antigenic activity of the peptides against AF.

1. Immobilization of IFA tablet.

On polystyrene plates (Costar, USA) was applied solutions of peptides in 0.01 M carbonate-phosphate buffer pH 7.4 at a concentration of 1 µg/100 µl. In each well of a tablet made of 100 μl of a solution. Sorption of the peptide was carried out at 2-4°C in accordance with is their 16 hours.

At the end of the sorption solution of peptide shook and each well was made a 2% solution of bovine serum albumin (BSA) (Sigma, USA). Incubation was performed for 2 hours at 37°C.

2. Analysis and detection of immune complexes (antigen-antibody).

The investigated samples were diluted 100 times with a solution of 2% BSA and the resulting solution was introduced into the wells at 100 μl. Incubated 1.5 hours at 37°C. after incubation, the tablet was washed three times with phosphate-saline with 0.05% tween-20 (SDF) 250 ál into the hole. Then for the formation of specific immune complexes in each well was made 100 ál of solution (1:1000) conjugate IgG rabbit antibodies to human IgG with horseradish peroxidase or protein a conjugate with horseradish peroxidase in 2% BSA and incubated for 1 hour at 37°C. After incubation, the solution was shaken off and the plate was washed three times with a solution of DCF. To implement the detection of immune complexes in each well was brought to 100 μl of TMB solution with hydroperiod and incubated in the dark at room temperature for 5-15 min to develop color. The reaction was stopped by adding to each well 50 µl of 50% hydrochloric acid. The colour intensity is measured on the vertical scanning photometer at 450 nm. Positive thought of serum at which the optical density (OD) was greater than the value of 0.2.

When tested immunohistochemistry its the STV peptides f-3 and f-4 showed high sensitivity AF (table 2, 3).

Table 2
Detection of antibodies to filaggrin (peptide f-3, f-4) in the sera of patients with RA
The total number ofPatients with AT to f-3Patients with AT to f-4
Patients of RA786673

When comparing immunochemical properties of f-3 and f-4 between themselves and with the properties already known active cyclic peptide CCP (HQCHQESTXGRSRCGRSGS, where X-Cit) was that there are patients in whom AF is defined by one of these peptides, while others do not. The highest sensitivity has f-4 (table 3, drawing). Based on these data, created antigenic complex for detection AF from a mixture of peptides (f-3; f-4).

Table 3
The data measure the optical density of the serum samples of donors and patients with RA
PatientsCCP(so450 nm)F-3(OP450 nm)F-4 (so450)
10,1470,2390,329of 0.3330,791
7 0,7430,3390,560,2830,588
12to 0.2630,2290,6410,2490,861
140,395 0,2150,1010,2430,291
170,16to 0.2630,310,2590,243
19of € 0.1950,2150,4370,2660,347
280,1580,180,516 0,3160,421
10,117has 0.1680,123is 0.135 0,08
D20,122is 0.135amount of 0.1180,1650,216
D60,1150,099is 0.1020,114-
D0.104 g0,1120,1160,119-
Note: in the column "patients" index "D" is marked serum donors.

1. The peptide having the amino acid sequence and structure chosen from:
where X is Cit, intended for the diagnosis of rheumatoid arthritis.

2. The peptide according to claim 1, immunological reactive immunological study aimed at the detection of rheumatoid arthritis.

3. A method for the diagnosis of rheumatoid arthritis, including the interaction of at least one of the peptides according to claim 1 or a mixture of peptides presented in claim 1, serum (plasma) blood of the patient and the diagnosis of rheumatoid arthritis by the presence of the formed complexes between the peptide and an antibody.

4. The method according to claim 3, in which the detection involves the use of enzyme-linked immunosorbent assay (ELISA).


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FIELD: medicine.

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6 cl, 1 ex, 2 tbl

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33 cl, 6 tbl, 30 ex

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13 cl, 3 tbl, 46 ex

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1 ex

FIELD: medicine.

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54 cl, 1 tbl, 9 dwg, 284 ex

FIELD: medicine.

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EFFECT: invention allows reducing or eliminating joint synovitis and oedema, downsising or softening ossificates in soft tissues, considerably lowering or arresting painful symptom, as well as reducing density and size of hematomas.

16 ex, 4 dwg, 4 cl

Pelotherapy // 2377030

FIELD: medicine.

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EFFECT: method improves clinical effectiveness ensured by higher activity of biological components of the mud.

7 cl, 4 ex

FIELD: medicine.

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3 ex

FIELD: medicine.

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EFFECT: method improves regenerative clinical effectiveness ensured maximum preservation of therapeutic properties of the prepared medical mud.

9 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention is related medicine and concerns applications of antibodies specifically recognising any prevailing variants of beta-amyloid peptide, Aβ40 and Aβ42, in preparation of a drug applied for prevention and-or treatment of Alzheimer's disease.

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7 cl, 3 ex, 2 dwg